CHAPITRE III
Implication thérapeutique du phénomène de tolérance l'ampicilline chez Hib â l'aide du modèle expérimental méningite chez les bb-rats.
L'importance clinique du phénomène de tolérance à l'ampicilline chez Hib est inconnue,
vivo et in vitro, les effets de
Nous avons étudié, in l'ampicilline avec trois souches d'Hi: une souche sensible (H395: CMB/CMI=
ng/ml), une souche tolérante (H34O: CMB/CMI = UQ/ml), et une souche
H g / m 1 ) .
résistante (H158: CMB/CMI=
0,25/0,25 32/0,125 1000/60
Pour les études a Hib chez des
in vivo, bb-rats le modèle expérimental a été utilisé. Cent-huit de méningite animaux ont reçu une unique
vingt-quatre heures dose après i.p. d'ampicilline l'induction i.p. de 200 mg/kg, l'infection. Pour les bactéries analysés
deux compartiments (sang et LCR), le taux de et la concentration d'antibiotique ont été â différents temps. Même si la concentration d'antibiotique dans le
animaux infectés avec
LCR était plus élevée la souche tolérante, tolérante a été
sensible mais un
inhibée plus lentement peu plus rapidement
que que chez les la souche la souche la souche résistante (p< 0.01).
Ces résultats démontrent pour la première fois, que phénomène de tolérance chez
traitement de la méningite
Hi exerce une influence â l'ampicilline.
dans le le
L'article ci-mentionné a été soumis au périodique Antimicrobial Agents and Chemotherapy.
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THERAPEUTIC SIGNIFICANCE OF AMPICILLIN TOLERANCE IN EXPERIMENTAL HAEMOPHILUS INFLUENZAE MENINGITIS.
Nathalie P. Tremblay, M.Sc.» Julie L'Ecuyer, B.Sc., Michel G. Bergeron, M.D. Service d'infectiologie, Le Centre Hospitalier de l'Université Laval et Département de microbiologie, Faculté de médecine, Université Laval, Québec, Canada.
Please address reprints to:
Dr. Michel G. Bergeron, MD FRCP Service d'infectiologie Le Centre Hospitalier de l'université Laval 2705 Boul. Laurier Ste-Foy, P.Q. G1V 4G2 Tel: (418) 654-2705
ABSTRACT
The signif icance of ampicillin tolérance in Haemophilus influenzae CHI) type b is unknown. We studied the in vivo and in vitro effects of ampicillin on three strains of Hl type b: a sensitive Hl (H395:MBC/MIC=0.25/0.25 ug/ml), a résistant Hl (H158:MBC/MIC-1OOO/6O|ig/ml), and a Hl (H340:MBC/MIC = 32/0.125 ug/ml). For the in vivo studies, the experimental model of Hl meningitis in infant rats was used. One hundred and eight animais were
hours
a single I.P. injection of 200mg/kg of ampicilin 24 after the induction of the infection. Antibiotic levels and bacterial counts were determined in CSF and blood at different intervals. Even though antibiotic levels with CSF were higher in the animais infected with the tolérant Hl in both the CSF and the blood, tolérant bacteria were inhibited more slowly than the sensitive strain (p<0.01), but only slightly more rapidly than the résistant strain (p<0.01). The findings provide the first substantial evidence that Hl tolérance exerts an influence on the response of meningitis to ampicillin treatment in vivo.
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INTRODUCTION
Bacterial meningitis continues to be major problem in infants and children. As many as 107. of these patients die and between 20-457. hâve severe sequelaes including
a
(deafness, blindness, seizures and mental retardation). The most common pathogens responsible for bacterial meningitis in infants and children are Haemophilus influenzae type b (Hib), Streptococcus pneumoniae and
Hib is the most important cause of bacterial meningitis in children between and 18 months.
Increasing level of résistance of Haemophilus influenzae has been observed since 1974. For the treatment of Haemophilus influenzae meningitis, the recommended regimen has been the
combination and chloramphénicol, but
cephalosporins céfotaxime, céfuroxime, and 3
ceftriaxone which hâve
and good CSF pénétration are now used as alternatives to the above combinations [1,2]. Although rare chloramphenicol résistance has been reported [3].
In 1981, we hâve reported influenzae may be tolérant phenomenon of tolérance
that s trains of Haemophilus to several beta-lactams. The of other microorganisms to antibiotics had been described previously [4,5]. In studies done to evaluate the clinicaal significance of tolérance, several investigators hâve estimated that tolérance could be associated with therapeutic failure, increase complications and mortality [5,9]. Although the clinical significance of tolérance in Hib is unknown, severe infections with organisms that are Killed slowly might be expected to respond more slowly to antimicrobial agents to which they are tolérant.
In the présent investigation we evaluated the
signif icance model of Hib
meningitis in infant rats.
MATE RI AL AND METHODS
BACTERIA Three strains of Haemophilus influenzae type
b were used. Strain H395 was sensitive to ampicillin (MBC/MIC:O.25/O.25u.g/ml),strain H158 was résistant to ampicillin (MBC/MIC:1000/60p,g/ml) while strain H34O was tolérant to ampicillin (MBC/MIC:32/0.125ng/ml). Ail of these strains were isolated f rom patients at the Centre Hospitalier de ^Université between 1975 and 1983 1). These strains were characterized (serotype, biotype, beta-lactamase production) as previously published [ 4 ] .
IN VITRO SUSCEPTIBILIT Y TESTING As previously reported.the
MBC and the MIC of ampicillin against the three strains were determined by a broth dilution susceptibility test. The assays were performed in triplicate with Mueller Hinton broth (Difco) supplemented with 1Z of hemin and 1/. of isovitalex (BBL Microbiology Systems) and ajusted to a pH of 7.4.
The in vitro growth and killing curves hâve been performed in broth using logarythmic and
The initial inoculum varied between
stationary phase 105-106 CFU per
inocula [10]. ml. The killing curves were done using a concentration of 2p./ml of ampicillin.
IN VIVO STUDIES MENINGITIS MODEL For these experiments, we hâve slightly modified the infant rat meningitis model of
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McColm [5,11]. Sprague-Dawley rats of 5-6 days of âge weighing between 8-14 g were used for the experiments.
Three strains of Hib were grown in supplemented Mueller Hinton broth (MHB) for 18 hrs at 37C and diluted to reach concentrations of 104 CFU per ml for the sensitive H395; 106 CFU per m) for the résistant H158 and tolérant H340. This smaller inoculum of the sensitive strain was used since preliminary experiments showed that
105x
with a higher
inoculum of or 106 CFU/ml,the animais given the sensitive H395 had a much higher log number CFU/ml of blood or CSF 24 hrs after the induction of the infection. After appropriate dilutions of HI, they were centrifuged at 5,000 rpm at 4C for 15 min and resuspended in and resuspended in a phosphate buffered saline containing 0.1Z gelatin (PBS-G) for a final volume of 5 ml Then, it was inoculated intraperitoneally in a volume of 0.1 ml. The percentage of bacteremia was 90Z and ail of the control animais ( 108 ) had a signif icant CSF involvement. Several controls including PBS-G ampicillin and untreated animais infected with each of the three strains of Hib were also used.
Ampicillin provided by Ayerst Laboratories was administered i.p. 24 hrs after infection. Groups of 6 animais were e valuated f rom 30 min to 24hrs f ollowing ampicillin injection. Ail of the infant rats were anesthetized i.p. with 0.5 ml of sodium pentobarbital (10mg/ml) before dissection CSF was collected from each animal by puncture of the cisterna magna. The skin and soft tissue over the cisterna magna were removed by dissection to expose the dura. The cisterna magna was entered by puncturing the dura with a stérile tuberculin syringe (1 cc 27 G 1/2). CSF f lowed outwards and could be drawn by capillary action into a 5 lilx 2 micropipets. Ail the CSF samples were clear. CSF red blood cells were evaluated with a Neubauer
hemocytometer; contamination with contained < 200 RBC/mm3. Blood
RBC was minimal and CSF samples were collected by cardiac puncture with a stérile tuberculin syringe. Ail the infant rats were then decapitated.
Rate of kill in vivo
colony counts af ter 24
eliminate the ail
saline containing O.2U of
Bacterial counts hrs incubation at
samples
were determined by 37°C in 5Z CO2. To were diluted with ase (Oxoid). The minimum limit of détection was 2x102 CFU/ml.
were performed
Whole using Bacillus subtilis as the were punched out
blood and an agar well microorganism. in the CSF antibiotic diffusion assay Wells of 2.0 agar using assays using mm a.d. a mini immunoelectrophoresis kit (Fisher). Standard curves were performed in blood for blood sample and buffered saline for CSF. The lower limit of dection was 0.2 p.g/ml.
Statistics The Bartlett and Duncan analyses were performed
to analyze the différence in killing of ampicillin with the t h r ee Haemophilus inf luenzae strains studied.
RESULTS
IN VITRO KILLING CURVES Results of susceptibility to
ampicillin of the three strains by broth dilution tests with stationary-phase inocula are presented in Fig. 1. It can be seen that the tolérant strain (H340) inhibited more than the sensitive strain (H395) but more rapidly than the résistant strain (H158) (P<0.01).
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RATE OF KILL IN VIVO: As shown in Fig.2 and 3, in both CSF
and blood the rate of killing of the tolérant strain was signif icantly slower than that of the sensitive strain (p<O.O1), but identical within its first eight hours to that of the résistant strain. Twenty-four hours after the administration of ampicillin we could still detect résistant bacteria (H158) while in blood and CSF of animais infected with the ampicillin tolérant strain we could not detect any bacteria.
PHARMACOKINETICS The pharmacokinetics of ampicillin in
normal animais and new born rats infected with the different strains of H. influenzae are compared in Table 2. In both CSF and blood ampicillin concentrations were observed to be higher in the infected animais than in the uninf ected animais (control). Of the 3 H influenzae, the p-lactamase to a lesser degree the producing strain seems to disturb
pharmacokinetic of ampicillin and, as observed in Fig. 4, the tolérant strain was associated with a marked increase in the AUC compared with the control and the two other H. inf luenzae (p<0.01). The ratio of concentration/MIC in both CSF and blood was much greater with tolérant strain.
DISCUSSION
Since 1974, isolâtes of HI hâve been shown to be résistant to ampicillin. The production of p-lactamase by these microorganisms has been responsible for this résistance. In the past years, we hâve observed that HI may also be tolérant to several beta-lactam antibiotics including third génération cephalosporins which are now used as alternatives
the toxic combina tion ampicillin-
chloramphenicol. The mechanism of this new form of résistance in H. inf luenzae is still unknown but this phenomenon has been well described in
bacter ia, par t icularly in Staphylococcus aureus and Streptococcus species [4,12]. We hâve thus undertaken experiments to see whether tolérance in H. inf luenzae c o u1 d t o
be of any clinical relevance, especially in the presence of meningitis where rapidly bactericidal agents are necessary to prevent severe complications.
The présent study investigated tolérance by using three strains of Hib (a tolérant, a sensitive and a résistant bacteria) in the experimental model of HI meningitis in infant rats. The rate of kill in vivo for ampicillin in infant rats dif f ered from those reported by other authors. For an identical initial inoculum, the number of CFU of Hib within blood and CSF after 24 hours infection were higher in our experiments than those reported by McColm et al [11]. Those investigators did not observe any différence in inf ected or uninf ected animais. Our observationi which shows that the ampicillin pharmacokinetics was different in the infected animais suggest that the meningitis might hâve been more severe.
We hâve no explanation for the marked variation in blood and CSF pharmacokinetics between the tolérant Hib and the other
strains, even though the This tolérant strain might induced septic shock and changes. We cannot exclude
peak sérum levels were identical. hâve been more virulent and rénal failure explaining these also the possibility of a more
f ind severe infection following H34O although we could not
any différence in the inflammatory response with the first 48 hours (unpublished data). In fact, as reported by Moxon et al, who hâve reported similar results [12], no inflammation of CSF is observed within the first 24 hrs of the induction of the meningitis.
The initial détectable number of
blood was almost identical, but within experiments it was évident
evaluated by the speed of
bacteria recovered in the CSF and the impressive within the susceptible strain CFU of Hib that the réduction of 4 in both CSF and hours of the clinical response the number of blood was more than with the fact, up tolérant and résistant microorganisms (P<0.01). In
to 8 hours the rate of killing of the tolérant and résistant strain was identical but, at 24 hrs, the résistant strain was still détectable in both humors The of ampicillin were even higher in the CSF and blood of animais infected with the tolérant strain, further suggesting that tolérance was responsible for this response to antibiotic. Some studies hâve shown that to hâve acceptable cure rates in meningitis the level of antibiotic in CSF must be at least eight to ten fold higher than the MIC. In the case of tolérant strain the ratio [14] blood or CSF antibiotic level over MIC the response to therapy was extremely slow. In contrast, the CSF levels were lower than the MBC suggesting that for meningitis one should arrive at levels way above the MBC to cure meningitis.
bacteria than H. inf luenzae hâve been done in an effort to A number of experimental studies using other tolérant
détermine whether this in vitro phenomenon is of signif icance in the treatment of infected patients. In experimental endocarditis, Goldman and Petersdorf found no signif icant différences between the responses of methicillin-tolerant and non-tolerant strains of S.aureus to prophylaxis or treatment [6]. Similar results were reported by Guze and al who compared the response of tolérant and non-tolerant Staphylococci to methicillin therapy in a rat of hematogenous pyelonephritis [7]. In contrast, Wilson and al found that tolérant Streptococci were killed rapidly by penicillin than non-tolerant organisme in rabbits with endocarditis [15].
The therapeutic implication of tolérance has not yet been delineated in humans or even in animais. As observed with several other microorganisms, the presence or absence of tolérance to antimicrobial agents is highly method dépendent. Factors that affect tolérance in vitro include the size of the inoculum used, the growth phase of bacteria, media, pH, or température of environment [16]. To validate the observations that were made in vitro [10], in vivo necessary to provide more
signif icance
As far as substantial influence on treatment in
information concer ning the possible of we ampicillin tolérance of are aware, evidence that the response Hib.
these findings provide the first
vivo. A slow or
tolérance exerts a criticial of HI infection to
with Hi meningitis to therapy suspect tolérance
inadéquate response with p-lactam
which is not limited to also been observed with third génération commonly used agents in Hib meningitis.
LITERATURE CITED of patients should make ampicillin but cephalosporins one has now
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1. Nelson John D. Emerging role of cephalosporins in
2. 3. 4. 5. 6. 7. 8. bacterial meningtis. 2A):47-51. Norrby S. Ragnar. treatment of Med. 1985;79 Moulin Wauters D., G. Haemophilus 144:259-260. Am. J. of Med. 1985; 79 (suppl. bacterial (suppl. Levecque cephalosporins meningitis in 2A):56-61. P., Mancilla, in the adults. Am. J. V.» PiOt, P., Fatal meningitis influenzae type due b. Brennan Robert significance of Streptococcal Chemother. McColm zidime against A.A., and meningitis.
O., Durack David penicillin tolérance endocar ditis. 1983;23:273-277. Ryan D.M. eleven J. to multi-resistant Eur. T. in J. 1985; Therapeutic experimental Antimicrob. Agents
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G. Significance experimental Staphylococcal endocarditis. Antimicrob. Agents Chemother. Guze Phyllis The rôle treatment aureus in Handwerger Of 1979;15:802-806.
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Ryan D.M. Evaluation of and chlor amphenicol in influenzae type b meningitis. Chemother. 1984;13:437-445.
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non-tolérant Staphylococcus methicillin treatment in an mice.
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Wilson W.R., Zak O., Sande M.A. Penicillin therapy for treatment of experimental endocarditis caused by viridans Streptococci in animais. J. Infect. Dis.
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16. Sherris John C. Problems in in vitro détermination of antobiotic tolérance in clinical isolâtes. Antimicrob. Agents Chemother. 1986;30:633-637.